1. Field of the Invention
The present invention generally relates to compositions and methods for in vitro culturing of a mammalian embryo and for enhancing achievement of pregnancy after implantation of the cultured embryo in the uterus of a suitable mammalian host.
2. Description of Related Art
Peroxisome proliferator activated receptor (PPAR) is a group of ligand activated transcription factors. It has three isotypes: PPARα, PPARγ, and PPARδ (also called PPARβ). The three PPARs belong to the nuclear receptor super family (Mangelsdorf et al., 1995), which includes steroid hormone receptor, thyroid hormone receptor, retinoid receptor and a growing number of orphan receptors. Ligand bound PPAR first heterodimerizes with RXR (which is a member of the retinoid receptor subfamily) before binding to the PPAR responsive element in the promoter of PPAR-responsive genes (Willson et al., 2000; Berger et al., 2002).
The roles of PPARα and PPARγ in energy homeostasis and inflammatory responses are suggested by their tissue-specific distribution and their natural ligands (Hihi et al., 2002; Wahli 2002). PPARα is present mainly in liver, brown adipose tissue and skeletal muscle; PPARγ is present mainly in the adipose tissue, macrophages and colon. Natural ligands for PPARα and PPARγ include polyunsaturated fatty acid (such as arachidonic acid and linoleic acid), leukotriene B4 (a product of arachidonic acid via lipoxygenase pathway), oxidized low-density lipoprotein, 9- and 13-hydroxoctadecadienoic acid, and, possibly, 15Δ-prostaglandin J2 (Forman et al., 1997). Synthetic PPARα and PPARγ ligands are used to treat lipid and glucose disorders: fibric acid, a PPARα ligand, is a lipid-lowering agent; thiazolidinedione, a PPARγ ligand, is an insulin sensitizer (Berger et al., 2002).
Although the identity of natural ligand(s) for PPARδ remains an enigma (Braissant et al., 1998), the diverse biological functions of PPARδ have been revealed by synthetic PPARδ ligand and mouse models with targeted PPARδ function (Peters et al., 2000; Barak et al., 2002). The reported functions of PPARδ include lipid homeostasis (Forman et al., 1997), endometrial receptivity (Lim et al., 2000; Ding et al., 2003), inflammation (Lee et al., 2003), wound healing (Michalik et al., 2001; Wahli 2002), myelination of the brain (Peters et al., 2000) and resistance to stress (Hao et al., 2002). Furthermore, PPARδ is implicated in colon cancer (Gupta et al., 2000; Cutler et al., 2003).
Prostacyclin (PGI2) is one of the putative natural ligands for PPARδ. High levels of PGI2 and PPARδ messages are coexpressed at implantation sites in the uterus (Lim et al., 1999). PGI2 analog or synthetic PPARδ ligand restores the loss of endometrial receptivity in cyclooxygenase-2 targeted mice (Lim et al., 1999; Lim et al., 2000). In renal medullary cells, which do not express PGI2 receptor, increased PGI2 production concomitant with enhanced activity of PPARδ response element promotes their survival during hypertonic stress (Hao et al., 2002). Finally, PGI2 is the possible link between non-steroidal anti-inflammatory drugs, which inhibit PG synthesis, and colon cancer prevention (Gupta et al., 2000; Cutler et al., 2003).
The development of preimplantation embryos in vivo is promoted by a coordinated program involving soluble factors from the oviducts and the uterus (Yeung et al., 1992). Prostacyclin (PGI2), one of the factors produced by the oviducts and the uterus via cyclooxygenase-2 (COX-2) pathway, plays a crucial role in embryo development and implementation (Huang et al., 2004a; Huang et al., 2004b; Huang et al., 2003; Lim et al., 1999). Compared with the development of in vivo embryos, the development of cultured embryos such as in vitro fertilized (IVF) embryos is retarded because cultured embryos were deprived of the protective environment of the oviduct (Hardy, 1997). Our recent work showed that supplementing culture media with iloprost, a stable analog of PGI2, enhances mouse blastocyst hatching in vitro (Huang et al., 2003). Furthermore, embryos preconditioned by iloprost show an enhanced potential of implantation and live births when transferred to gestational carriers (Huang et al., 2004b). A source of PGI2, other than the oviducts and uterus, is preimplantation embryos. Blocking the production of endogenous PGI2 by selective COX-2 inhibitors retards embryo hatching (Huang et al., 2004c). Thus, PGI2 is crucial for embryo development in vitro, and its stable analog effectively improves the hatching and implantation of cultured embryos. Although preimplantation embryos express PGI2 receptor and possess functional protein kinase A, PGI2 analog did not increase their cAMP levels (Huang et al., 2003). It remains unclear how PGI2 and its analogs achieve these enhancements.
PGI2 exerts its effects by binding to a G-protein coupled prostaglandin receptor (IP) and/or peroxisome proliferator-activated receptor δ (PPARδ) (Forman et al., 1997; Namba et al., 1994). The inhibition of platelet aggregation and the relaxation of smooth muscle cells by PGI2 are mediated by IP receptors via the cyclic AMP-dependent kinase (PKA) pathway (Namba et al., 1994). PPARδ has been implicated in cell protection by PGI2 (Adderley and Fitzgerald, 1999; Hao et al., 2002; Tan et al., 2001). IP null mice have increased propensity for thrombosis (Cheng et al., 2002); PPARδ null mice exhibit reproductive defects (Barak et al., 2002; Cheng et al., 2002).
It has been previously reported that embryos cultured in medium supplemented with PGI2 analog showed enhanced hatching (Huang et al., 2003), implantation and live birth (Huang et al., 2004; International Patent Application PCT/US2004/029167 (Huang et al.) entitled “Method and Composition for Enhancing In Vitro Embryo Development By Supplementing Culture Medium with Prostaglandin or a Prostaglandin Analog”; and U.S. patent application Ser. No. 11/370,152 (Huang et al.) entitled “Enhancement of Mammalian Embryo Development,” the disclosures of which are hereby incorporated herein by reference. Ways to further increase IVF success by improving in vitro development of preimplantation embryos to enhance their potential for being successful implanted in utero, and to increase the rate of live births from such embryos continue to be sought.